Available Online JOURNAL OF SCIENTIFIC RESEARCH Publications J. Sci. Res. 3 (2), 403-411 (2011) www.banglajol.info/index.php/JSR Study on Isolation and Identification of Salmonella and Escherichia coli from Different Poultry Feeds of Savar Region of Dhaka, Bangladesh A. Chowdhuri1, A. Iqbal1*, M. Giasuddin2, and A. A. Bhuiyan2 1Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and Technology, Sylhet-3114, Bangladesh 2Bangladesh Livestock Research Institute, Savar, Dhaka, Bangladesh Received 18 February 2011, accepted in final revised form 11 April 2011 Abstract The study was conducted aiming at the isolation and identification of Salmonella and Escherichia coli (E. coli) from different brands of poultry feeds sold in Savar, Dhaka, Bangladesh. Seven different poultry feeds were subjected to microbiological analysis. All these samples were analyzed by culturing in different media such as nutrient broth (NB), nutrient Agar (NA), SS Agar (Salmonella-Shigella Agar), BGA (brilliant-green Agar), Mac Conkey, DHL and EMB (eosin methylene blue) media. Total bacterial colonies of all the samples were counted separately on the nutrient Agar media. Hence, bacteria were counted to be 9.5×105 in the feed sample C (Layer) which was found to be the highest in number among the poultry feeds. Total viable count (TVC) of Salmonella and E. coli in the feed samples were as 0 to 6.75×104 and 0 to 3.05×104 respectively. Both organisms were found in 71.43% and 57.14% of the analyzed feed samples, respectively. The highest number of Salmonella was found in sample C (Layer) feeds and that of E. coli was found in sample B (Grower) feeds. The widespread occurrence of Salmonella and E. coli in poultry feeds reinforces the need for effective control measures, hygiene in processing and handling of feeds. Keywords: Salmonella; Escherichia coli; Poultry feeds; Total viable count; Contamination; Hygiene. © 2011 JSR Publications. ISSN: 2070-0237 (Print); 2070-0245 (Online). All rights reserved. doi:10.3329/jsr.v3i2.7128 J. Sci. Res. 3 (2), 403-411 (2011) 1. Introduction Poultry feeds are food materials used in raising poultry birds. Poultry feeds are referred to as complete feeds as they are designed to contain all the nutritional materials needed for proper growth, meat and egg production in birds. Various brands of poultry feeds are in existence depending on the functions they perform in the birds. Thus, there are growers, finishers, layers, starters among others. Poultry feeds can potentially become contaminated with food borne pathogenic microorganisms during harvesting and eventual * Corresponding author: [email protected] 404 Study on Isolation marketing of the bagged feeds. Poultry feeds contaminated with bacteria pathogenic to humans can contribute to human food borne illness through the feed-poultry-food-human chain. The production of poultry feeds requires microbiological safety regulations to escape microbial contamination of the product. Prominent among these microorganisms, the bacteria Salmonella and E. coli infections of poultry have been shown to be of critical importance in Bangladesh. Salmonella are spread from poultry to humans, often through foods such as eggs and meat. Salmonella spp. causes an intestinal infection in humans known as Salmonellosis [1]. The investigation of survey of Salmonella serovars in broilers and laying breeding reproducers in Eastern Algeria was conducted [2]. One egg colonized with Salmonella could contaminate all eggs and chicks during hatching [3]. Salmonella contamination of food products can significantly reduce consumer demand and affect producer profits [4]. E. coli are one of other common microbial floras of gastrointestinal tract of poultry [5, 6]. Among the diseases some are often severe and sometimes lethal infections such as meningitis, endocarditis, urinary tract infection, septicemia, epidemic diarrhoea of adults and children [7] and yolk sac infection, omphalitis, cellulitis, swollen head syndrome, coligranuloma and colibacillosisn [8]. Enteritis caused by E. coli (colibacilliosis) is an important disease in the poultry industry because of increased mortality and decreased performance [9, 10]. Therefore, the objectives of the present study is to investigate (i) the isolation and identification E. coli and Salmonella strains from different sources of poultry feeds, (ii) the prevalence and enumeration of E. coli and Salmonella from different sources of poultry feeds and (iii) the causes of contamination of bacterial load in the poultry feeds. The current study was conducted at Bangladesh Livestock Research Institute (BLRI), Savar, Dhaka, Bangladesh aiming at the microbial assessment of the various brands of poultry feeds available at Savar market, Dhaka. 2. Materials and Methods 2.1. Study area The present study was carried out at the Animal Health Research Division (AHRD), BLRI, Savar, Dhaka during the period from April 2010 to June 2010. 2.2. Collection of samples Seven different brand types of poultry feed samples were aseptically collected from the different poultry farm and poultry market places at Savar, Dhaka, Bangladesh. These samples of different brands were labeled by Sample A (Grower), Sample B (Grower), Sample C (Layer), Sample D (Starter), Sample E (Starter), Sample F (Layer), Sample G (Starter) and taken immediately to the laboratory of BLRI, Savar, Dhaka. A. Chowdhuri et al., J. Sci. Res. 3 (2), 403-411 (2011) 405 2.3. Bacteriological analysis The samples were analyzed within 2-6 hours of collection. The different media such as nutrient Agar (NA), nutrient Broth (NB), SS Agar (Salmonella-Shigella Agar), BGA (Brilliant Green Agar), EMB (eosin methylene blue), Mc Conkey and DHL were prepared separately. The last five media are called selective media. The above media were prepared separately by the following method: 2.3.1. Preparation of nutrient Agar (NA) media The Nutrient Agar media were prepared by suspending 14gm nutrient Agar in 500ml distilled water in a bicker and boiled to dissolve completely. The media and some Petridis were sterilized by autoclaving at 121oC for 20 minutes at 15lbs. Then media were kept on the Petridis sterilizing by laminar air flow. 2.3.2. Preparation of nutrient broth (NB) media The Nutrient Broth media were prepared by suspending 6.5 gm nutrient broth in 500ml distilled water. The media were heated to dissolve completely. The media were sterilized by autoclaving at 121oC for 20 minutes at 15lbs pressure. Then media were kept on the Petridis sterilizing by laminar air flow. 2.3.3. Preparation of SS Agar media The SS Agar media were prepared by suspending 31.5 gm SS Agar in 500ml distilled water. The media were heated to boiling with frequent agitation to dissolve completely but not autoclaved or overheated, because overheating may destroy the selectivity of the medium. The media were cooled to about 50oc. The media were mixed well and poured into sterile Petridis sterilizing by laminar air flow. 2.3.4. Preparation of BGA media The BGA Agar media were prepared by suspending 29gm BGA Agar in 500ml distilled water. The media were heated to dissolve completely and sterilized by autoclaving at 121oC for 20 minutes at 15lbs pressure. Overheating was avoided for more selectivity; aseptically added rehydrated contents of 1 vial of sulpha supplement (FDO68). The media were mixed well before pouring into sterile Petridis. 2.3.5. Preparation of Mc Conkey media The media were prepared by suspending 27.75 gm Mac Conkey Agar in 500ml distilled water. The media were heated to boiling with gentle swirling to dissolve completely. The 406 Study on Isolation media were sterilized by autoclaving at 121oC for 20 minutes at 15lbs pressure. Overheating was avoided. Then media were cooled to 45-50oC and poured into sterile Petridis. The surface of the medium was dried when inoculated. 2.3.6. Preparation of EMB Agar media The media were prepared by suspending 18 gm EMB Agar in 500ml distilled water. The media were heated to boiling to dissolve completely. The media were sterilized by autoclaving at 121oC for 20 minutes at 15lbs pressure. Overheating was avoided and was cooled to 50oC. Then the medium was shaken in order to oxidize the methylene blue (i.e., to restore its blue color) and to suspend the flocculent precipitate. The samples were first cultured into the nonselective media such as nutrient Agar and nutrient broth media for total bacterial count. Then these samples were subcultured into the selective media for identification of the bacteria by their colony morphology. Again the samples were direct cultured to the selective media for enumeration of the total identified bacteria. For culturing, 10 gm feed samples were taken and then ground. Then 90 ml peptone water was poured into the bicker and mixed with the samples. Then 900 l PBS was taken to each of the small bottles accordingly. Then 100 l mix sample from bicker was taken to the one of the small bottles for serial dilution. Thus the serial dilution wasμ made up to 10-4. These appropriate dilutions were cultured by spreadμ plate technique using sterile bent glass rod on the NA media. These inoculated NA media were then incubated overnight at 37oC in the incubator. Thus serial dilution of other samples were done by the same way and incubated overnight at 37oC. Then the bacteria of different samples were grown and formed many colonies to the NA media. Then these colonies were counted which is called Total viable count (TVC). On the other hand, all the feed samples were measured in 1 gm and taken to the NB media in the test tube separately and kept in the incubator at 37oC overnight. For sub culturing, the colonies of the NA media were inoculated in the selective media by looping for the identification of Salmonella and E. coli bacteria from the different feed samples and were incubated at 37oC overnight. On the other hand, the samples of the NB media were inoculated to all the selective media by looping from the different feed samples.